Using stable isotope labeling/mass spectrometry to probe Cdk1 multisite phosphory

描述(申请人提供)：拟议的试点项目旨在开发一种定量质谱学方法，以详细和受控的方式研究多位点磷酸化，并将该方法应用于细胞周期蛋白依赖的激酶底物的多位点磷酸化。由于缺乏能够在明确条件下定量分析磷酸化动力学的技术，多位点磷酸化的详细研究一直受到阻碍。在这里，稳定的同位素标记和质谱联用将被应用于发芽酵母细胞周期蛋白依赖性激酶CDK1和一些底物分子之间的体外磷酸化反应。稳定同位素标记/质谱学是一种成熟的技术，具有适当的灵敏度、动态范围和精确度，用于以时间分辨、特定部位的方式进行准确量化蛋白质磷酸化所需的测量。已发表和未发表的初步研究表明，CDK1可以从酵母细胞中以活性形式分离出来，并可用于体外磷酸化反应。这一建议旨在将多位点CDK1磷酸化理解为生物物理系统，因此需要体外动力学研究提供的精确控制。体外方法是首选的，因为在复杂的体内细胞环境中总是存在隐藏的变量，例如相关酶之间的串扰和单一培养中不同蛋白质群体的存在。具体地说，这项建议旨在确定CDK1磷酸化的过程性或分布性；以及位点的顺序和依赖性(如果有的话)被磷酸化。此外，CDK1在体内有9个潜在的细胞周期蛋白结合伙伴/激活物，存在于细胞周期的不同点，具有部分重叠的特异性和功能。这些多位点磷酸化研究的结果可能有助于深入了解细胞周期蛋白和细胞周期蛋白特异性模式之间的差异。试点项目的一个主要部分还将是通过与一位非常成功的导师合作，传播研究成果，建立合作关系，并最终获得未来的资金，促进国际和平研究所的专业发展。与公众健康相关：这项提案旨在利用前沿分析技术来了解细胞分裂的主要参与者--细胞周期蛋白依赖性激酶的活性。从拟议的真核生物模型酿酒酵母研究中获得的知识将对阐明原理以及人类生物学，特别是与细胞增殖和癌症调控有关的原理具有价值。提出的创新的体外磷酸化/定量质谱学方法也有助于研究其他多部位酶系统，如糖原合成酶和酪蛋白激酶-与代谢调节、炎症、阿尔茨海默病和其他神经疾病等过程密切相关的蛋白质。

DESCRIPTION (provided by applicant): The proposed pilot project intends to develop a quantitative mass spectrometric method for studying multisite phosphorylation in a detailed and controlled manner, and to apply that method to the multisite phosphorylation of cyclin-dependent kinase substrates. The detailed study of multisite phosphorylation has been hampered by the lack of techniques capable of quantitatively analyzing phosphorylation dynamics under well-defined conditions. Here, stable isotope-labeling coupled to mass spectrometry will be applied to in vitro phosphorylation reactions between the budding yeast cyclin-dependent kinase Cdk1 and a number of substrate molecules. Stable isotope-labeling/mass spectrometry is a well-established technique with the appropriate sensitivity, dynamic range and precision for making the measurements that will be required to accurately quantify protein phosphorylation in a time-resolved, site-specific manner. Published and unpublished preliminary studies have been performed to show that Cdk1 can be isolated from yeast cells in an active form, and can be used for in vitro phosphorylation reactions. This proposal aims to understand multisite Cdk1 phosphorylation as a biophysical system, and therefore requires the precise control afforded by in vitro kinetic studies. The in vitro approach is preferred because hidden variables are invariably present in the complicated in vivo cellular milieu, such as cross-talk between related enzymes and the presence of diverse protein populations within a single culture. Specifically, this proposal aims to determine the processivity or distributivity of Cdk1 phosphorylation; and the order and dependence, if any, of sites are phosphorylated. Furthermore, Cdk1 has 9 potential in vivo cyclin binding partner/activators present at different points in cell cycle that have partially overlapping specificity and function. Results of these multisite phosphorylation studies may shed insight into differences between the cyclins and modes of cyclin specificity. A major part of the pilot project will also be the professional development of the PI through collaboration with a highly successful mentor, research dissemination, establishment of collaborations, and ultimately, the acquisition of future funding. RELEVANCE TO PUBLIC HEALTH: This proposal aims to utilize leading-edge analytical technology to understand the activity of cyclin dependent kinases, a main player in cell division. Knowledge gained from the proposed study on the model eukaryote S. cerevisiae will have value in elucidating principles that also human biology, particularly as it relates to regulation of cell proliferation and cancer. The innovative in vitro phosphorylation/quantitative mass spectrometry approach proposed also lends itself readily to studying other multisite enzyme systems, such as the glycogen synthase kinases, and casein kinases-proteins that are intimately involved in processes such as metabolic regulation, inflammation, and Alzheimer's disease and other neurological disorders.